Proper replication of the DNA is critical for passing on of the genetic information when a cell divides. Alterations to the DNA code, which occur as a result of both intrinsic and extrinsic DNA damage, can cause gene mutations leading to alterations in protein function and expression. This may ultimately lead to disease states such as cancer. Therefore, the cellular response to replication stress is important for maintaining the genomic integrity and health of an organism. The replication stress response is complex and requires the coordination of a number of cellular pathways, including cell cycle checkpoint, replication fork and replisome stabilization, prevention and restart of replication fork re-firing, DNA damage repair, and restart of DNA replication. Due to the intricacy of these pathways, understanding of these responses is an ongoing process in which new players are being discovered regularly. We believe a number of novel proteins associated with the replication stress response are yet to be identified. Consequently, in Aim 1 we propose to use a whole genome siRNA approach in order to identify proteins that play a role in replication stress responses. An immunofluorescence assay utilizing thymidine analog incorporation and the replication stress inducer hydroxyurea has been designed to uncover proteins that are necessary for replication stress repair and replication restart. This assay takes advantage of the 384-well plate format and high-throughput analysis of immunofluorescent data in order to quickly identify proteins of interest. Once proteins of interest are validated, they will be furthe characterized in Aim 2 to classify them and prioritize their function in the replication stress response. Classification and prioritization will be based upon bioinformatics analyses and experimental evaluation to determine sensitivity to hydroxyurea and replication stress, subcellular localization of the protein, effect on DNA replication and replication fork dynamics, as well as determination of whether the protein is involved in an ATR-dependent or -independent pathway. These analyses will allow for identification of high- priority proteins playing roles in the replication stress response. Further studies will focus on determining the mechanisms by which these proteins function during replication stress. The proposed aims will allow for identification of novel replication stress response proteins that will deepen our understanding of DNA replication and repair and how these pathways contribute to genomic stability. A greater appreciation for the proteins and mechanisms that function in genomic stability will ultimately lead to improved developments in cancer diagnostics and therapeutics.